Fig 1.
Gene structures and temporal expression patterns of Spo and Jhamt during embryogenesis.
(A) Schematics of Spo (red) and Jhamt (blue) genes. Coding sequences (CDSs) are shown in dark-colored boxes and untranslated regions (UTRs) are indicated in light-colored boxes. siRNA target sites are denoted by black triangles. Scale bar, 250 nt. (B) Expression of Spo (red) and Jhamt (blue) during embryogenesis quantified by qRT-PCR. Timing of hatching and molting to instar 1 juveniles are shown under the x-axis. Expression levels are normalized to those of the reference gene ribosomal L32. Values are means. Error bars represent SD (N = 3). ***p<0.001 (Student’s t-test).
Fig 2.
Spo is essential during early embryogenesis in Daphnia magna.
(A) Developmental stages of D. magna during early embryogenesis at 23°C as observed by GFP fluorescence in an H2B-GFP transgenic line. (B) Expression levels of Spo in siControl and siSpo embryos at 6 h assessed by qRT-PCR. Values are means. Error bars represent SD (N = 3). **p<0.01 (Student’s t-test). (C) Developmental progression and survival rate of siControl and siSpo with or without exposure to 1 μM 20-hydroxyecdysone (20E). Bars denote the developmental stages described in (A): stage 1 (white), stage 2 (blue), stage 3 (green), stage 4 (yellow), stage 5 (gray). (D) Representative images of embryos injected with siControl, siSpo, and siSpo rescued by 20E. Embryos were imaged by GFP fluorescence. Representative photographs from different stages are combined into a single image. (E) Retardation of embryo developmental progression. Compound eye (a) and naupliar eye (b) development observed at 46 h after ovulation. Scale bar, 200 μm.
Fig 3.
Jhamt knockdown shortens Daphnia magna embryonic period.
(A) Embryonic periods (hours after ovulation, or hao) of siControl, siJhamt, and siMet embryos with or without fenoxycarb. *p<0.05; **p<0.01; ***p<0.001 (Kruskal-Wallis followed by Dunn post-hoc test; p-values adjusted using the Benjamini-Hochberg procedure). (B) Embryonic period of embryos exposed to different concentrations of fenoxycarb. (C) Expression levels of Jhamt in 24 h embryos injected with siControl and siJhamt as measured by qRT-PCR. Values are means. Error bars represent SD (N = 3). **p<0.01 (Student’s t-test).
Table 1.
RNAi inhibition of sesquiterpenoid pathway genes.
Fig 4.
Jhamt knockdown upregulates expression of ecdysteroid biosynthesis genes at 24 hao.
(A) Predicted ecdysteroid biosynthesis pathway in Daphnia magna. C: cholesterol; 7-dC: 7-dehydrocholesterol; 5-KD: 5β-ketodiol; 5-KT: 5β-ketotriol; 2dE: 2-deoxyecdysone; E: ecdysone; 20E: 20-hydroxyecdysone. Nvd: Neverland; Spo: Spook; Phm: Phantom; Dib: Disembodied; Sad: Shadow; Shd: Shade. (B) Expression of Nvd2, Spo, Dib, and Cyp18a1 at 24 h in siControl, siJhamt and siMet embryos at 24 h after siRNAt as measured by qRT-PCR. (C) Expression levels of other ecdysteroidogenesis genes in siControl, siJhamt, and siMet embryos. Expression levels are normalized to those of ribosomal L32, then shown relative to expression levels of siRNA-ctrl. Values are means. Error bars represent SD (N = 3). *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).
Fig 5.
Schematic diagram of proposed ecdysteroid and sesquiterpenoid cross-talk in Daphnia magna embryo.
Solid lines indicate direct interactions whereas dashed line indicates possible interaction. Sesquiterpenoid through sesquiterpenoid signaling cascade downregulates ecdysteroidogenesis gene expression, hence suggesting antagonistic relationship between sesquiterpenoid and ecdysteroid.